Small-conductance calcium (Ca2+)-activated potassium (SK) channels are activated solely by intracellular Ca2+ and underlie the after hyperpolarization (AHP) and firing frequency in central neurons and other excitable cells. Due to their functional diversity, SK channels are potential targets for drug development in the treatment of a number of different diseases, and these efforts would be greatly enhanced by a deeper understanding of their activation mechanism. SK channels rely on the ubiquitous Ca2+ binding protein, calmodulin (CaM), which associates with the channel constitutively to sense changes in intracellular Ca2+. A critical step to understanding the Ca2+-activation of SK channels hinges on obtaining separate measurements of Ca2+ binding and channel opening. Through these measurements, the contribution of Ca2+ binding steps to opening can be determined. Here we propose to measure Ca2+ binding and channel activation simultaneously in functioning SK channels using a combined optical and electrophysiological approach. Additionally, we propose to fully characterize Ca2+ binding to CaM through measures of EF hand-specific affinities and the degree of binding cooperativity. Luminescent, trivalent lanthanide ions (Ln3+) can effectively substitute for spectroscopically silent Ca2+ in activating SK channels. Eu3+ and Tb3+ can be directly excited, or Tb3+ luminescence can be sensitized through energy transfer from an adjacent aromatic amino acid. By introducing an energy-transferring residue into the Ca2+ binding domain, the latter method allows for site-specific monitoring of Tb3+ binding. Using direct Ln3+ excitation, though site specificity is lost, binding is distinguished through an increase in the Ln3+ luminescence decay lifetime, and measurements can utilize wild-type CaM. In either case, the measured luminescence intensity is directly proportional to the fraction of bound Ln3+ and is therefore a direct measurement of the binding isotherm. The experiments presented in this proposal are the first to directly measure the site-specific Ca2+ and Ln3+ binding affinities of individual EF hands in both lobes of intact CaM. Additionally, the cooperativity of Ca2+ binding will be assessed using mutations that significantly reduce Ca2+ binding affinity adjacent EF hands. The combination of site-specific measurements of Ca2+ affinities and direct measurements of the degree of binding cooperativity will aid in the development of a quantitative understanding of Ca2+ binding to CaM. Additionally, the direct measurement of Ca2+ binding to functioning SK channels in membrane patches is only the second of its kind in an ion channel. The development of this tool certainly has large implications for improving our understanding of the Ca2+-activation of the SK channel, however, the proposed method is also widely applicable to numerous channels that are modulated by CaM. PUBLIC HEALTH RELEVANCE: Ion channels, such as the small-conductance calcium-activated potassium (SK) channel, are important drug targets for a variety of diseases. The development of efficient and effective drugs that target SK channels would benefit greatly from a detailed understanding of their activation mechanism and function. The work proposed here will investigate the calcium activation of SK channels using a novel approach that can also be applied to a number of other ion channels that are modulated by calcium.